Fixatives Flashcards
What is the difference between tolerant and non tolerant?
Tolerant:
- Tissues can be exposed for a long time without adverse effects.
Non-Tolerant:
- Tissues cannot be exposed for a long time without adverse effects.
What is a primary fixing agent?
A single chemical that has the ability to denature protein
How are primary fixatives typically used?
Almost never used alone; usually combined in a compound fixative
What are some common primary fixatives?
- Formaldehyde
- Potassium dichromate
- Mercuric chloride
- Ethanol
- Picric acid
- Acetic acid
- Osmium tetroxide
- Glutaraldehyde
What is formaldehyde commonly known as?
The universal fixative
What is the typical concentration of formalin used as a routine fixative?
10% vol/vol, which contains 4% formaldehyde
What type of fixative is formaldehyde considered?
An additive fixing agent
What is the main reaction site for formaldehyde in tissues?
Amino group (NH2) found on the side chains of amino acids
What is formed when formaldehyde reacts with tissue proteins?
A cross-linkage called a methylene bridge
True or False: Formaldehyde causes more shrinkage than any other fixative.
False
What happens to glycogen when fixed with formaldehyde?
Glycogen is indirectly fixed; trapped in a meshwork
What effect does formaldehyde have on lipids?
Lipids are preserved but not made insoluble
How can formalin pigment be formed?
When formaldehyde is used in acidic solutions (pH < 5)
What happens to glycogen when fixed with potassium dichromate?
Glycogen is oxidized and not readily demonstrable
What is the effect of potassium dichromate on tissue pH?
It is pH dependent; non-coagulating at pH > 3.8
What is a disadvantage of potassium dichromate?
Highly toxic and considered a carcinogen
What is mercuric chloride known for?
Powerful protein coagulant and extremely toxic
How does mercuric chloride affect lipids?
Has no reaction with lipids; does not fix or destroy them
What must be done to remove mercury pigment from tissues?
Treat with metallic iodine followed by sodium thiosulfate
What effect does mercuric chloride have on tissues if over-exposed?
Excessive hardening, making tissues brittle
What is the mode of action of mercuric chloride?
Combines with acid groups of proteins and inhibits freezing
What is the procedure for mercury pigment removal from tissue?
- Immerse in alcoholic iodine for 3 minutes.
- Wash in running tap water for 2 minutes.
- Immerse in 5% HYPO.
- Wash in running tap water for 2 minutes.
- Stain as required.
This procedure effectively removes mercury and iodine discolouration.
What is a key characteristic of mercuric chloride in histological applications?
It is never used alone and always combined with gentler solutions.
Mercuric chloride is typically part of compound fixatives.
True or False: Ethanol is a good fixing agent for demonstrating microanatomy.
False
Ethanol causes extreme shrinkage, making it a poor choice for microanatomy demonstration.
What does ethanol best preserve?
Glycogen
Ethanol precipitates glycogen, allowing it to be demonstrated in tissue sections.
What type of fixative is picric acid classified as?
Additive coagulant fixative
Picric acid forms additive compounds with proteins.
What is a major disadvantage of picric acid?
Causes extreme shrinkage and penetrates poorly
These properties limit its effectiveness in some applications.
How does acetic acid affect the nuclei during fixation?
Coagulating and intolerant
Acetic acid causes significant swelling and affects nucleoproteins.
What is the main use of osmium tetroxide in histology?
Fixation for electron microscopy
Osmium tetroxide preserves fine cellular structures.
What type of fixative is glutaraldehyde?
Non-coagulating additive fixative
Glutaraldehyde crosslinks proteins and is used primarily for electron microscopy.
What is a disadvantage of glutaraldehyde?
Slow penetration and must be stored in a refrigerator
These factors affect its usability in tissue processing.
Fill in the blank: Picric acid is used as both a _______ and a dye.
fixative
Picric acid serves dual purposes in histological applications.
True or False: Acetic acid preserves lipids in tissues.
False
Acetic acid does not fix or destroy lipids; they are removed during processing.
What happens to glycogen when fixed with glutaraldehyde?
Glycogen proteins are precipitated in a meshwork
This indirect fixation allows glycogen to be demonstrated with specific stains.
What is glyoxal used for in histology?
Replacing glutaraldehyde and formaldehyde
Glyoxal is less toxic and does not give off vapors.
What are the primary fixing agents used in compound fixatives?
- Neutral buffered formalin
- Formal saline
- Formal calcium
- Buffered formal sucrose
- Formal alcohol
- Zenker’s
- Bouin’s
- B – 5
- Zinc containing
What is the purpose of combining fixing agents in a compound fixative?
To balance/counteract the harmful effects of one fixative with the beneficial effects of another.
Which agent is commonly used to counteract shrinkage in a compound fixative?
Acetic acid.
What is the function of salts or buffers in a compound fixative?
- Maintain osmotic pressure
- Aid in penetration of fixative into tissues.
What are the categories into which Compound Fixatives are subdivided?
- Microanatomical
- Cytological
- Histochemical.
What are the components of Neutral Buffered Formalin?
- Formaldehyde
- Phosphate buffer
- Water.
What is a disadvantage of Neutral Buffered Formalin?
Gradual loss of basophilic staining of cytoplasm and nucleus after prolonged storage.
What is the stock solution of Zenker’s Fluid composed of?
- Mercuric chloride
- Potassium dichromate
- Sodium sulphate
- Distilled water
- Glacial acetic acid.
What is a disadvantage of Zenker’s Acetic Fixative?
Turns muddy brown after 24 hours.
What is the purpose of acetic acid in Zenker’s Acetic Fixative?
Acts as a primary fixative and excellent nucleoprotein fixative, counteracts shrinkage.
What are the components of Bouin’s Fixative?
- Saturated picric acid
- Formalin
- Glacial acetic acid.
What is the primary use of Bouin’s Fixative?
Good general all-purpose fixative, excellent for preserving soft and delicate structures.
What are the disadvantages of Bouin’s Fixative?
- Lyses RBCs
- Alters and decreases lipids
- Tissues must be transferred directly to alcohol to prevent loss of water soluble picrate.
What are the ingredients of B – 5 Fixative?
- Mercuric chloride
- Sodium acetate
- Distilled water
- Formalin (added immediately prior to use).
What tissues is B – 5 Fixative used to fix?
Hematopoietic and lymphoreticular tissues.
What are the components of Z – 5 Fixative?
- Zinc sulphate
- Unbuffered formalin
- Ethanol.
What is the composition of Carnoy’s Fixative?
- Absolute ethanol
- Chloroform
- Glacial acetic acid.
What is the unique feature of Carnoy’s Fixative?
The only compound fixative classified as a cytological fixative.
How quickly does Carnoy’s Fixative penetrate tissue?
Rapid penetration for the first 2mm of tissue.
What type of fixative is Carnoy’s classified as?
Carnoy’s is classified as a cytological fixative.
How long does it take for a 2mm tissue to be fixed using Carnoy’s?
A 2mm tissue will be fixed in 15 minutes.
What advantage does Carnoy’s fixation provide for urgent diagnosis?
Rapid fixation at room temperature allows for STAT or urgent diagnosis.
What components does Carnoy’s fluid excellently preserve?
Carnoy’s gives excellent nuclear fixation and preserves nucleic acids.
What substance is an important ingredient in fixing glycogen?
Alcohol is an important ingredient to fix glycogen.
What are the disadvantages of using Carnoy’s fluid?
Carnoy’s fluid causes considerable shrinkage and destroys or dissolves most cytoplasmic elements.
What happens to lipids when using Carnoy’s fluid?
Lipids are dissolved by ethanol.
What effect does acetic acid have on RBCs in Carnoy’s fluid?
RBCs are lysed by acetic acid.
What is the purpose of histochemical fixatives?
Histochemical fixatives preserve chemical constituents in or on cells.
What is often lost when using histochemical fixatives?
The architecture of the tissue and cellular morphology are often forfeited.
What is a secondary fixative?
A secondary fixative is an additional fixative used after the first fixative.
What improvement does secondary fixation provide?
Shows general improvement in preservation of tissue.
What is the primary fixative usually utilized?
NBF (Neutral Buffered Formalin).
What are common components of secondary fixatives?
- Mercury
- Picric acid
What is the purpose of mordanting qualities in fixatives?
Mordanting properties enhance or improve subsequent staining of the tissue.
What is mercuric chloride used for?
Often used as a secondary fixative.
What is Bouin’s solution used for?
Used as a mordant prior to staining tissue sections with the trichrome staining method.
What is the recommended ratio of fixative to tissue for proper fixation?
20:1.
What can result from inadequate fixative volume?
Poor fixation.
